PCT application WO 95/09866 published 13 Apr. 1995 and incorporated herein by reference, discloses a group of GST-activated compounds which rely on interaction of a prodrug form of a drug or toxin with glutathione S-transferase and the resulting abstraction of a proton by the enzyme, releasing an electron pair which mediates, in turn, the release of the drug or toxin. These compounds are generally of the following formula, where the pathway of released electrons from hydrogen ion abstraction is indicated. ##STR1## In these compounds, L is an electron withdrawing leaving group;
S.sup.x is an oxidized form of S, Se or C, e.g., S.dbd.O, O.dbd.S.dbd.O, S.dbd.NH, HN.dbd.S.dbd.O, Se.dbd.O, O.dbd.Se.dbd.O, Se.dbd.NH, HN.dbd.Se.dbd.O, S.sup.+ R' wherein R' is alkyl (1-6C) or O--C.dbd.O or HN--C.dbd.O; PA1 each R is independently H or a noninterfering substituent; PA1 n is 0, 1 or 2, PA1 YCO is selected from the group consisting of .gamma.-Glu, .beta.-Asp, Glu, Asp, .gamma.-GluGly, .beta.-AspGly, GluGly and AspGly; and PA1 AA.sub.C is an amino acid linked through a peptide bond to the remainder of the compound. PA1 selecting from said panel an analog which interacts with said MRP system; PA1 synthesizing a prodrug which is a conjugate of the appropriate form of the selected analog with a substance of the desired biological activity; and PA1 administering the resulting prodrug to a subject in need of treatment with the biologically active compound. PA1 YCO is selected from the group consisting of .gamma.-Glu, .beta.-Asp, Glu, Asp, .gamma.-GluGly, .beta.-AspGly, GluGly and AspGly; and PA1 AA.sub.C is an amino acid linked through a peptide bond to the remainder of said compound of formula (1); PA1 (conj) is a conjugated system permitting transfer of electron pairs, such as --CR.dbd.CR--; --(CR.dbd.CR).sub.2 -- or --phenylene--; PA1 m is 0 or 1; each R is independently H or noninterfering substituent; PA1 the dotted lines represent alternative covalent bonds tethering the biomolecule to the indicated C; and PA1 "biomolecule" represents a moiety which is biologically active when covalent bond (a) is severed. PA1 each X is independently O, NH or S; PA1 Z is a moiety which, when associated with P(O)X.sup.1 X.sup.2 is biologically active; and PA1 the dotted lines represent alternative covalent bonds linking CR.sub.2 and thus X.sup.2 to the remainder of the molecule--i.e., to C*, C.sup.+, or a carbon in the conjugated system if present.
As explained in the above-cited PCT application, specificity with respect to particular tissues or targets can be manipulated, mainly through appropriate choices for AA.sub.C, and to a lesser extent, YCO. The reason for this is that the nature of the glutathione analog portion of the prodrug determines which of the many isozymes of GST are effective in releasing the biologically active moiety. The nature of the leaving group will determine the biological effect of administering the prodrug. Included among the leaving groups described are nitrogen mustards and other cytotoxic substances, as well as various antibiotics, indicator molecules, and other groups.
Although the prodrugs described in the PCT application are effective, they may also be cleared more quickly than desired from the target cells or tissue by virtue of elevated levels of multidrug resistance associated protein (MRP) which transports GSH-conjugated substances out of the cell as described by Jedlitschky, G. et al., Cancer Research (1994) 54:4833. For example, in the case of the phosphoramide mustards, displacement of a chloride ion from one of the 2-chloroethyl groups by the sulfhydryl group of glutathione results in a GSH-conjugate which can then be cleared by the MRP system.
It would be desirable to provide prodrugs which not only release active forms of the drugs per se, but also result in a lowered rate of clearance of the activated drug. The present invention provides two approaches to this problem. One approach resides in selecting, as the glutathione analog in the prodrug, a glutathione analog that itself interacts with the MRP, e.g., in competition with GSH. Thus, after the prodrug is cleaved by GST, the glutathione analog can inhibit the transport of other moieties, such as the activated drug or toxin. This approach is workable, however, only where the specificity desired for the prodrug release permits this choice to be made.
In a more universal approach, the prodrug is designed to activate, but not to release completely the biologically active moiety associated with it; the biologically active moiety remains tethered to the glutathione analog, reducing its susceptibility to GST-mediated conjugation to free GSH. Thus, it has reduced ability to form a compound which is effectively cleared by the MRP system.